While erythropoietin (Epo) is commonly used to treat anemia in patients with cancer and kidney disease, recent studies have raised major safety concerns. There is great interest in developing new strategies to modulate erythropoiesis, and in this regard, we have been analyzing GATA transcription factor function. GATA-2 regulates the proliferation/survival of hematopoietic stem cells (HSCs), whereas GATA-1 promotes the differentiation of erythroid, megakaryocyte, eosinophil, and mast cells. Analysis of GATA-1 function in GATA-1-null erythroid precursor cells revealed a novel target gene, mFam55B. GATA-1 directly activates mFam55B transcription, and mFam55B expression is highly restricted to definitive erythroblasts in murine fetal liver and adult bone marrow. Although mFam55B has minor sequence similarity to Intercellular Cell Adhesion Molecule-4 (ICAM4), which is implicated in formation of functionally important erythroblastic islands, the bulk of mFam55B sequence does not resemble ICAM4 or other known proteins. mFam55B appears to be a Type II membrane protein, whereas ICAMs are Type I membrane proteins. We discovered four mammalian mFam55B-related genes, which have not been described, but only mFam55B is GATA-1-regulated. mFam55B is predicted to have a transmembrane helix and two extracellular immunoglobulin (Ig)-like repeats. mFam55B and its paralogs define a new protein family, and we hypothesize that mFam55B is an erythroid precursor membrane protein with important functions in erythroid cell biology. The following aims describe pilot/exploratory studies to develop reagents/models to define mFam55B function and to investigate the role of mFam55B in the formation of erythroblastic islands. Aim 1 - To determine whether mFam55B is a GATA-1-regulated transmembrane protein in erythroid precursor cells. We hypothesize that mFam55B is a transmembrane protein in erythroid precursor cells that functions via intercellular signaling. We will test whether GATA-1 regulates endogenous mFam55B levels, whether mFam55B exhibits predominant plasma membrane localization, and whether it resides in erythroblastic islands. Aim 2 - To investigate biological functions of mFam55B via loss-of-function studies in zebrafish and mice. We identified five novel zebrafish genes with significant sequence similarity to mFam55B. Knocking down one of these genes yielded severe anemia. We will knockdown the mFam55B-related genes to determine if they regulate erythropoiesis, will define their expression patterns, and will test whether their expression is GATA-1-dependent. We will also generate a mFam55B-null mouse to begin to dissect the role of mFam55B in erythroid cell function and/or erythropoiesis. PUBLIC HEALTH RELEVANCE: This project focuses on analyzing a novel GATA-1 regulated protein, mFam55B, that we discovered, which is predicted to be a single-pass transmembrane protein on erythroid precursor cells and to represent the founding member of a new protein family. As GATA-1 is a fundamental regulator of erythropoiesis, we hypothesize that mFam55B has important functions to regulate red blood cell development and/or erythroid precursor cell function. The proposed pilot/exploratory studies therefore have potential to yield high impact findings of relevance to novel strategies to stimulate red blood cell development, to elucidating how erythroid precursor cell function is altered in pathophysiological states, and to unraveling fundamental aspects of protein structure/function.